The long term objectives of my research is to determine the mechanisms that control the initiation of protein synthesis in yeast. The goal of this proposal is to describe in genetic and molecular terms the functional relationship between the 5' end of the HIS4 transcript and the position of the AUG initiator codon, and its specific interactions with the components of the translational initiation complex. The following specific aims are directed at achieving this goal: 1) Using special selective properties of the HIS4 locus I have devised an in vivo genetic selection scheme that enables the direct selection of mutations that reduce or abolish initiation at the normal AUG start codon. This scheme should identify flanking nucleotide sequences that serve to direct ribosomal recognition of the initiator codon. 2) Using recent advances in recombinant DNA technology mutations will be constructed in vitro throughout the 5' non-coding region of the HIS4 transcript and put back at site to study their in vivo effects. Deletion and insertion mutations will enable us to address sequence and structural complexities in this region that may not be realized by in vivo selections. 3) Both in vivo and in vitro constructed mutations will then be used in reversion tests which restore the ability to express HIS4. A novel selection scheme is presented that should enable us to easily distinguish between internal and external suppressors. Internal suppressors may identify other important sequences in the initiator region; external suppressors should define components of the translational initiation complex that have altered specificities for the mutant HIS4 initiator regions. This work should be directly related to many studies in higher eukaryotes contributing valuable information into the basic control mechanisms that determine proper and efficient gene expression. Furthermore, the genetic identification of the components of the translational initiation complex will enable us to construct probes for these genes that will allow a comprehensive analysis of the expression and regulation of their essential gene products. A study of this type may provide important insight into mechanisms that coordinate normal cellular growth in yeast.